These experiments are designed to provide the detailed functional and structural analysis of neuronal circuit organization in the colon that is crucial for the mechanisms of reflex control of colonic function to be determined. This study will determine the origins, projections and terminations of different chemically-defined classes of neuron in the colon of the guinea pig. The distributions within neurons and their processes of peptides including dynorphin, enkephalin, galanin, neuromedin U, neuropeptide Y, somatostatin, substance P and vasoactive intestinal peptide, of the neuronal marker calbindin D28K and of the putative transmitters serotonin and gamma-aminobutyric acid will be determined using immunocytochemical methods. Pathways will be determined by analyzing patterns of fiber degeneration after nerve lesions. The co-localization of substances in neurons will be determined by simultaneous double-labelling immunocytochemistry. Combined intracellular recording techniques and immunocytochemistry will be used to correlate the neurochemistry and morphology of neurons with their functions. Responses from enteric ganglion cells and muscle cells of the circular muscle layer will be recorded with intracellular microelectrodes. The neurons will be characterized by their biophysical properties and synaptic inputs. Fluorescent dye will be injected through the recording electrode to mark neurons and determine their shape. The tissue will then be fixed and stained to reveal the chemistry of each categorized neuron. In order to determine the roles of particular neurons, two types of reflex stimulus will be used: circumferential stretch and mechanical distortion of the mucosa. These stimuli will be used to evaluate the places of categorized neurons in specific circuits mediating stereotyped reflexes. The pathways will be further analyzed by recording transmission from motor neurons to the circular muscle at various distances from the mechanical stimuli. These responses will be analyzed in terms of amplitudes and durations, responses to drugs affecting ganglionic transmission, and after specific nerve lesions. This project should culminate in the production for the first time of structurally and functionally validated neuronal circuit maps for the control of the colon. This knowledge will be of considerable value in understanding how neural control of colonic function is mediated and is necessary information for adequate understanding of disorders of colonic control mechanisms.